Compounds and methods for treating cancer

ABSTRACT

The present application provides methods and compositions for treating cancer, for example, renal cell carcinoma, melanoma, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, and thyroid cancer, and more particularly to using an inhibitor of a stearoyl-Coenzyme A desaturase 1 (SCD1) enzyme in combination with a checkpoint inhibitor to treat these disorders.

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.16/489,133, filed Aug. 27, 2019, which is a National Stage Applicationunder 35 U.S.C. § 371 that claims the benefit of application Serial No.PCT/US2018/020257, filed Feb. 28, 2018, which also claims the benefit ofU.S. Provisional Application Ser. No. 62/465,062, filed Feb. 28, 2017.The disclosures of the prior applications are considered part of (andare incorporated by reference in) the disclosure of this application.

TECHNICAL FIELD

This document relates to methods and compositions for treating cancer,for example, renal cell carcinoma, melanoma, ovarian, breast, prostate,colon, pancreatic, bladder, liver, lung, and thyroid cancer, and moreparticularly to using one or more inhibitors of a stearoyl-Coenzyme Adesaturase 1 (SCD1) enzyme in combination with one or more checkpointinhibitors to treat these disorders.

BACKGROUND

SCD1 is an enzyme that catalyzes the de novo lipogenesis of 4-9monounsaturated fatty acids (MUFA) oleic acid (OA) and palmitoleic acid(PA). These MUFAs are essential for the synthesis of triglycerides,sphingolipids, ceramides, glycolipids, phospholipids, and otherlipoproteins which influence membrane fluidity, membrane raft formationand receptor clustering, second messenger signaling, fatty acidoxidation, energy storage, cell division, inflammation, and a number ofother biological functions. SCD1 has been implicated as pro-tumorigenicin a multitude of cancers, such as clear cell renal cell carcinoma(ccRCC).

SUMMARY

Agents that specifically target crucial metabolic enzymes utilized bycancer have been actively investigated. However, it is unclear whetherinhibition of fatty acid metabolism in tumors affects theirimmunogenicity. The present application shows that inhibition, e.g., ofstearoyl-CoA desaturase 1 (SCD1), a key enzyme involved in fatty-acidsynthesis and a potential prognostic marker for human cancers, increasesthe immunogenic susceptibility of cells and tumors, e.g., poorlyimmunogenic tumors. Inhibition of SCD1 can increase both recruitment andactivation of immune cells in vivo, which when combined with PD-1blockade can result in potent and durable anti-tumor T cell responses.Inhibition of tumorigenic de novo lipogenesis represents a novelapproach to enhance T cell based cancer immunotherapy such as checkpointinhibitor therapy.

In a first general aspect, the present application provides a method oftreating cancer in a subject, the method comprising administering to thesubject a therapeutically effective amount of an inhibitor of de novolipogenesis, or a pharmaceutically acceptable salt thereof, and atherapeutically effective amount of a checkpoint inhibitor, or apharmaceutically acceptable salt thereof.

In some embodiments, the checkpoint inhibitor is a programmed cell deathprotein-1 (PD-1) inhibitor.

In some embodiments, the checkpoint inhibitor is an inhibitor ofprogrammed death-ligand-1 (PD-L1) or programmed death-ligand-2 (PD-L2).

In some embodiments, the checkpoint inhibitor is an inhibitor ofcytotoxic T-lymphocyte-associated protein-4 (CTLA-4).

In some embodiments, the checkpoint inhibitor is an inhibitor of T-cellimmunoglobulin and mucin-domain containing-3 (TIM-3).

In some embodiments, the checkpoint inhibitor is an antibody.

In some embodiments, the antibody is monoclonal.

In some embodiments, the checkpoint inhibitor is pembrolizumab.

In some embodiments, the checkpoint inhibitor is nivolumab.

In some embodiments, the checkpoint inhibitor is atezolizumab.

In some embodiments, the checkpoint inhibitor is ipilimumab.

In some embodiments, the checkpoint inhibitor is TSR-022.

In some embodiments, the inhibitor of de novo lipogenesis isadministered with at least two checkpoint inhibitors.

In some embodiments, the at least two checkpoint inhibitors are selectedfrom the group consisting of: pembrolizumab, nivolumab, atezolizumab,ipilimumab, and TSR-022.

In some embodiments, the inhibitor of de novo lipogenesis isadministered with at least three checkpoint inhibitors.

In some embodiments, the at least three checkpoint inhibitors areselected from the group consisting of: pembrolizumab, nivolumab,atezolizumab, ipilimumab, and TSR-022.

In some embodiments, the inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof, and the checkpoint inhibitor,or a pharmaceutically acceptable salt thereof, are admixed prior toadministration.

In some embodiments, the inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof, and the checkpoint inhibitor,or a pharmaceutically acceptable salt thereof, are administeredconcurrently.

In some embodiments, the inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof, and the checkpoint inhibitor,or a pharmaceutically acceptable salt thereof, are administeredsequentially. In some embodiments, the inhibitor of de novo lipogenesis,or a pharmaceutically acceptable salt thereof, is administered prior tothe administration of the checkpoint inhibitor, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the checkpoint inhibitor, or a pharmaceuticallyacceptable salt thereof, is administered prior to the administration ofthe inhibitor of de novo lipogenesis, or a pharmaceutically acceptablesalt thereof.

In some embodiments, the inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof, is administered orally.

In some embodiments, the checkpoint inhibitor, or a pharmaceuticallyacceptable salt thereof, is administered intravenously.

In some embodiments, the inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof, is administered in an amountfrom about 200 mg/kg to about 250 mg/kg.

In some embodiments, the checkpoint inhibitor, or a pharmaceuticallyacceptable salt thereof, is administered in an amount from about 1 mg/kgto about 15 mg/kg.

In some embodiments, the molar ratio of the inhibitor of de novolipogenesis, or a pharmaceutically acceptable salt thereof, to thecheckpoint inhibitor, or a pharmaceutically acceptable salt thereof, isfrom about 150:1 to about 1:3.

In some embodiments, the cancer is selected from the group consistingof: a kidney cancer, a liver cancer, a breast cancer, a lung cancer, apancreatic cancer, a bladder cancer, a colon cancer, a melanoma, athyroid cancer, an ovarian cancer, and a prostate cancer.

In some embodiments, the cancer is breast cancer.

In some embodiments, the breast cancer is HER2-positive breast cancer.

In some embodiments, the cancer is clear cell renal cell carcinoma(ccRCC).

In some embodiments, the cancer is a kidney cancer.

In some embodiments, the cancer is a bladder cancer.

In some embodiments, the bladder cancer is selected from the groupconsisting of: transitional cell carcinoma, urothelial carcinoma,papillary carcinoma, flat carcinoma, squamous cell carcinoma,adenocarcinoma, small-cell carcinoma, and sarcoma.

In some embodiments, the bladder cancer is a transitional cellcarcinoma.

In some embodiments, the cancer is a thyroid cancer.

In some embodiments, the cancer is a liver cancer.

In some embodiments, the liver cancer is hepatobiliary carcinoma (HCC).

In some embodiments, the cancer is a lung cancer.

In some embodiments, the lung cancer is non-small cell lung cancer(NSCLC).

In some embodiments, the cancer is a solid tumor.

In a second general aspect, the present application provides a method ofincreasing the immunogenic susceptibility of a cell, the methodcomprising: i) selecting a poorly immunogenic cell; and ii) contactingthe cell with an effective amount of an inhibitor of de novolipogenesis, or a pharmaceutically acceptable salt thereof.

In some embodiments, the cell is a cancer cell.

In some embodiments, the cancer is selected from the group consistingof: a kidney cancer, a liver cancer, a breast cancer, a lung cancer, apancreatic cancer, a bladder cancer, a colon cancer, a melanoma, athyroid cancer, an ovarian cancer, and a prostate cancer.

In some embodiments, the cancer is breast cancer.

In some embodiments, the breast cancer is HER2-positive breast cancer.

In some embodiments, the cancer is clear cell renal cell carcinoma(ccRCC).

In some embodiments, the cancer is a kidney cancer.

In some embodiments, the cancer is a bladder cancer.

In some embodiments, the bladder cancer is selected from the groupconsisting of: transitional cell carcinoma, urothelial carcinoma,papillary carcinoma, flat carcinoma, squamous cell carcinoma,adenocarcinoma, small-cell carcinoma, and sarcoma.

In some embodiments, the bladder cancer is a transitional cellcarcinoma.

In some embodiments, the cancer is a thyroid cancer.

In some embodiments, the cancer is a liver cancer.

In some embodiments, the liver cancer is hepatobiliary carcinoma (HCC).

In some embodiments, the cancer is a solid tumor.

In some embodiments, the contacting is in vitro.

In some embodiments, the contacting is in vivo.

In some embodiments, the cell becomes susceptible to cell lysis inducedby an immune cell.

In some embodiments, the immune cell is a T lymphocyte.

In some embodiments, the T lymphocyte is CD4+ T-cell.

In some embodiments, the T lymphocyte is CD8+ T-cell.

In a third general aspect, the present application provides method ofincreasing the immunogenic susceptibility of a tumor in a subject, themethod comprising: i) selecting a subject having a poorly immunogenictumor; and ii) administering to the subject a therapeutically effectiveamount of an inhibitor of de novo lipogenesis, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the tumor is cancerous.

The method of claim 62, wherein the tumor is selected from the groupconsisting of: a kidney cancer, a liver cancer, a breast cancer, a lungcancer, a pancreatic cancer, a bladder cancer, a colon cancer, amelanoma, a thyroid cancer, an ovarian cancer, and a prostate cancer.

In some embodiments, the tumor is breast cancer.

In some embodiments, the breast cancer is HER2-positive breast cancer.

In some embodiments, the tumor is clear cell renal cell carcinoma(ccRCC).

In some embodiments, the tumor is a kidney cancer.

In some embodiments, the tumor is a bladder cancer.

In some embodiments, the bladder cancer is selected from the groupconsisting of: transitional cell carcinoma, urothelial carcinoma,papillary carcinoma, flat carcinoma, squamous cell carcinoma,adenocarcinoma, small-cell carcinoma, and sarcoma.

In some embodiments, the bladder cancer is a transitional cellcarcinoma.

In some embodiments, the tumor is a thyroid cancer.

In some embodiments, the tumor is a liver cancer.

In some embodiments, the liver cancer is hepatobiliary carcinoma (HCC).

In some embodiments, the tumor is solid.

In some embodiments, the subject is a mammal.

In some embodiments, the subject is a human.

In some embodiments, the inhibitor of de novo lipogenesis isadministered orally.

In some embodiments, the inhibitor of de novo lipogenesis isadministered in an amount from about 200 mg/kg to about 250 mg/kg.

In some embodiments, the cells of the tumor become susceptible to celllysis induced by an immune cell.

In some embodiments, the immune cell is a T lymphocyte.

In some embodiments, the T lymphocyte is CD4+ T-cell.

In some embodiments, the T lymphocyte is CD8+ T-cell.

In some embodiments, increasing the immunogenic susceptibility of thetumor sensitizes the tumor for immunotherapy or a checkpoint inhibitortherapy.

Implementations of the first, second, and third general aspects mayinclude one or more of the following features.

In some embodiments, the inhibitor of de novo lipogenesis is theinhibitor of fatty-acid synthesis.

In some embodiments, the fatty acid is a Δ-9 monounsaturated fatty acid(MUFA).

In some embodiments, the MUFA is oleic acid (OA).

In some embodiments, the MUFA is palmitoleic acid (PA).

In some embodiments, the inhibitor of de novo lipogenesis is theinhibitor of stearoyl-CoA desaturase 1 (SCD1).

In some embodiments, the inhibitor of de novo lipogenesis is a compoundof Formula (I):

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein:    -   R¹ is an unsubstituted C₁₋₆alkyl or C₁₋₆haloalkyl;    -   X is

-   -   Y is selected from the group consisting of:

-   -   m is 0 or 1;    -   n is 0, 1, or 2;    -   V is NR⁴ or O;    -   R², R³, and R⁴ are each independently H or an unsubstituted        C₁₋₆alkyl; and    -   Z is an unsubstituted aryl.

In some embodiments, the compound according to Formula (I) has thestructure of Formula (Ia):

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is CF₃.

In some embodiments, m is 0.

In some embodiments, V is NH.

In some embodiments, Y is

In some embodiments, Y is

In some embodiments, m is 1.

In some embodiments, V is O.

In some embodiments, Y is

In some embodiments, R² is H; and R³ is CH₃.

In some embodiments, Y is

In some embodiments, n is 1.

In some embodiments, Z is phenyl.

In some embodiments, the compound according to Formula (I) is selectedfrom the group consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of de novo lipogenesis is a compoundof Formula (II):

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein    -   R¹ is halo;    -   X is —(C═O)NR⁴—;    -   Y is

and

-   -   R², R³, and R⁴ are each independently H or an unsubstituted        C₁₋₆alkyl.

In some embodiments, the compound according to Formula (II) has thestructure of Formula (IIa):

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is Cl.

In some embodiments, R⁴ is H.

In some embodiments, R² is H; and R³ is CH₃.

In some embodiments, the compound according to Formula (II) is

or a pharmaceutically acceptable salt thereof.

In some embodiments, the method of treating cancer in a subjectcomprises administering to the subject a therapeutically effectiveamount of:

or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of a PD-1 inhibitor, or a pharmaceutically acceptablesalt thereof.

In some embodiments, the PD-1 inhibitor is pembrolizumab or nivolumab.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present application belongs. Methods and materialsare described herein for use in the present application; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the present application will beapparent from the following detailed description and figures, and fromthe claims.

DESCRIPTION OF DRAWINGS

FIG. 1A shows gene array and ancillary pathway signature analysis ofSCD1 inhibitor treated ccRCC cells.

FIG. 1B shows maximal SCD1 over-expression in HER2 enriched breastcancer.

FIG. 1C shows that elevated SCD1 mRNA expression correlates with reducedoverall survival in all breast cancer patients.

FIG. 1D shows that elevated SCD1 mRNA expression correlates with reducedoverall survival in Her2-enriched breast cancer patients.

FIG. 1E shows increased levels of endoplasmic reticulum stress responsefactors including binding immunoglobulin protein (BiP, GRP78), totalphosphorylated eukaryotic translation initiation factor 2A (eIF2A) atserine51 and DNA damage inducible transcript 3 (CHOP, DDIT3) in TUBO,E0771-E2, and MMTV-neu tumor cells.

FIG. 1F shows results of flow cytometry on live tumor cells following a48 hour treatment with SSI-4 at 10 nM, demonstrating that SSI-4 inducesplasma membrane translocation of calreticulin, a known immunogenic celldeath inducer.

FIG. 2A shows that SSI-4 (1000 nM) induced the highest level ofphagocytosis in both TUBO and E0771-E2 cells (5 and 13%, respectively),and this effect is reversed in with adjuvant CRT neutralization.

FIG. 2B shows that SSI-4 treatment (1000 nM) significantly enhancedantigen presentation in both TUBO (8.5%) and E0771-E2 (5%) cells, andthat this effect was inhibited with adjuvant CRT neutralization.

FIG. 2C shows that SSI-4 treatment (1000 nM) significantly enhanced OT-ICD8 T cell proliferation in both E0771-E2 and MMTV-neu cells, and thatthis effect was inhibited with adjuvant CRT neutralization in MMTV-neucells. This effect was dependent on the co-presence of macrophages (MP),T cells (T) and tumor cells.

FIG. 2D shows that SSI-4 treatment (1000 nM) significantly enhancedinterferon gamma (IFNγ) production in OT-I CD8 T cells in both E0771-E2and MMTV-neu cells, and that this effect was inhibited with adjuvant CRTneutralization in MMTV-neu cells. This effect was dependent on theco-presence of macrophages (MP), T cells (T) and tumor cells.

FIG. 3A shows that SSI-4 treated animals demonstrated slower tumorprogression, with markedly smaller tumor sizes recorded at 30 days afteronset of therapy when control animals reached endpoint.

FIG. 3B shows an appreciable increase in overall survival in SSI-4treated mice.

FIG. 3C shows that H&E staining of tumor sections did not revealconspicuous changes in overall tissue morphology between sham and SSI-4treatment groups.

FIG. 3D shows a significant decrease in Ki-67 protein staining,indicative of decreased tumor proliferation was noted in the SSI-4group. (%+nuclei=47.7±6.4 for sham, %+nuclei=36.7±6.2 for SSI-4).

FIG. 3E shows a significant increase in cleaved caspase 3 (CC3)staining, indicative of enhanced tumor apoptosis was noted in the SSI-4group. (H=4.9±2.6 for sham, H=10.7±3.9 for SSI-4).

FIG. 3F shows 10% increase in the number of tumor-associated leukocyteswithin SSI-4 treated animals.

FIG. 3G shows that SSI-4 treated tumors demonstrated increasedintra-tumor penetration of macrophages, identified by positive F4/80 IHCstaining. (% pop.=31.7±4.1, % pop.=43.3±8.6).

FIG. 3H shows a significant increase in the number of intra-tumordendritic cells within SSI-4 treated tumors as compared to both controltreated tumors, and normal mammary tissue from non-tumor bearing mice.

FIG. 3I shows that macrophages isolated from SSI-4 treated tumorsdemonstrate increased expression of pro-inflammatory cytokines, anddecreased expression of immunosuppressive cytokines as compared tocontrol treated tumors, and normal mammary tissue from non-tumor bearingmice.

FIG. 4A shows an increase in the number of CD4+ tumor-infiltratingpopulations in SSI-4 treated tumors.

FIG. 4B shows an increase in the number of CD8+ tumor-infiltratingpopulations in SSI-4 treated tumors.

FIG. 4C shows an increase in perforin in SSI-4 treated tumors.

FIG. 4D shows that SSI-4 treatment produced a robust induction of memoryand effector T-cells among CD4 positive T cell populations.

FIG. 4E shows that SSI-4 treatment produced a robust induction of memoryand effector T-cells among CD8 positive T cell populations.

FIG. 4F shows that effector CD4 and CD8 T lymphocytes identified by IFNγwas markedly increased in both CD4 and CD8+ T cell populations fromSSI-4 treated tumors. SSI-4 also significantly reduced the number ofintra-tumor CD4+ T regulatory cells identified by dual CD25 and FoxP3expression.

FIG. 4G shows that SSI-4 treatment induces programmed death ligand-1(PD-L1) expression in TUBO tumor bearing mice.

FIG. 5A Is an example treatment schedule for mice receiving combinationtherapy including SSI-4 and PD-1 checkpoint blockade.

FIGS. 5B-5C show that EO771-E2 tumors do not respond to monotherapeuticPD-1 blockade; combination of PD-1 blockade with SSI-4 produced a moredurable anti-tumor response (5B); median survival in the combinationgroup increased by approximately 45% compared to both placebo and PD-1alone, and 20% as compared to SSI-4 monotherapy.

FIGS. 5D-5E show an increase in effector CD8+ cytotoxic T lymphocytes inresponse to SSI-4 and combination therapy.

FIG. 5F shows that PD-1 monotherapy and combination therapy had adeleterious effect on the intratumor population of T regulatory cells.

FIG. 5G shows that depletion of CD8 T lymphocytes rescued the anti-tumoractivity of the combination treatment.

FIG. 5H shows splenic depletion of CD8 T-cells in animals receiving CD-8blockade.

FIG. 6A shows that macrophages are the predominant resident leukocyte instudied tumors, and both SSI-4 and combination therapy increasedintratumor infiltration of macrophages by approximately 8% and 5%,respectively.

FIG. 6B shows that no significant changes in the protein expression ofthe checkpoints PD-L1 or PD-L2 were observed in dendritic cells.

FIG. 6C shows that macrophages showed increased PD-L1 in response toboth SSI-4 and combination therapy.

FIGS. 6D-6E show that T lymphocytes demonstrate upregulation of proteinexpression of various checkpoints in response to therapy, includingCTLA-4 and TIM3.

FIG. 7 shows results of flow cytometry on live tumor cells(HER2-positive and TNBC breast cancer) following a 48 hour treatmentwith SSI-4 (10-1000 nM), demonstrating that SSI-4 induces plasmamembrane translocation of calreticulin, a known immunogenic cell deathinducer. Significance shown for 1000 nM dose (anova).

FIG. 8 shows that SSI-4 (1000 nM) induced the highest level ofphagocytosis of both TUBO and E0771-E2 HER2-positive breast cancer cells(5 and 13%, respectively), and this effect is reversed in with adjuvantCRT neutralization.

FIG. 9 shows that SSI-4 (1000 nM) induced the highest level ofphagocytosis of both 4T1 and E0771 TNBC breast cancer cells (12.5 and9.4%, respectively), and this effect is reversed in with adjuvant CRTneutralization.

FIG. 10 shows that SSI-4 treatment (1000 nM) significantly enhancedantigen presentation in both TUBO (8.5%) and E0771-E2 (5%) cells, andthat this effect was inhibited with adjuvant CRT neutralization.

FIG. 11 shows that SSI-4 treatment (1000 nM) significantly enhanced OT-ICD8 T cell proliferation in both E0771-E2 and MMTV-neu cells, and thatthis effect was inhibited with adjuvant CRT neutralization.

FIG. 12 shows that SSI-4 treatment (1000 nM) significantly enhancedOT-II CD4 T cell proliferation in both E0771-E2 and MMTV-neu cells, andthat this effect was inhibited with adjuvant CRT neutralization.

FIG. 13 T cell proliferation in FIG. 2D-F was dependent on theco-presence of macrophages (BMDM), T cells (T), and tumor cells as SSI-4treated T cells alone and T cells plus macrophages could not induce Tcell proliferation.

FIG. 14 shows T cell proliferation in response to SSI-4 treatment inFIG. 2D-E is antigen-dependent, as expression of cOVA antigen by tumorcells was required, as those bearing empty vector (EV) could notsimilarly stimulate T cell proliferation.

FIG. 15 shows an appreciable increase in overall survival in SSI-4treated mice. H&E staining of tumor sections did not reveal conspicuouschanges in overall tissue morphology between sham and SSI-4 treatmentgroups.

FIG. 16 shows a significant increase in the number of intra-tumordendritic cells within SSI-4 treated tumors as compared to both controltreated tumors, and normal mammary tissue from non-tumor bearing mice.

FIG. 17 shows an increase in the number of CD4+ tumor-infiltratingpopulations in SSI-4 treated tumors.

FIG. 18 shows an increase in the number of CD8+ tumor-infiltratingpopulations in SSI-4 treated tumors. Of note, CD8 T cells were presentboth peripherally and centrally in SSI-4 treated tumors, whereas thoseseen in control tumors were predominantly peripheral.

FIG. 19 shows that SSI-4 treatment produced a robust induction ofsplenic memory (T_(CM)) and effector (TEFF) T-cells among CD4 positive Tcell populations.

FIG. 20 shows that SSI-4 treatment produced a robust induction ofsplenic memory and effector T-cells among CD8 positive T cellpopulations.

FIG. 21 shows that effector CD4 and CD8 T lymphocytes identified by IFNγwas markedly increased in both CD4 and CD8+ T cell populations fromSSI-4 treated TUBO tumors. SSI-4 also significantly reduced the numberof intra-tumor CD4+T regulatory cells identified by dual CD25 and FoxP3expression.

FIG. 22 shows that SSI-4 treatment induces programmed death ligand-1(PD-L1) 5 expression in both TUBO and E0771-E2 HER2-positive breasttumor bearing mice.

FIGS. 23A-23B show that TUBO HER2-positive tumors do not respond tomonotherapeutic PD1 blockade; combination of PD-1 blockade with SSI-4produced a more durable anti-tumor response; Complete tumor regressionand durable survival achieved in 83% of combination treated animals,where all animals in other treatment groups succumbed to tumor burden.SSI-4 monotherapy treated animals demonstrated improved median survivalof approximately 37% and 35% as compared to sham and PD-1 monotherapytreated groups, respectively.

FIGS. 23C-23D show that EO771-E2 HER2-positive tumors do not respond tomonotherapeutic PD1 blockade; combination of PD-1 blockade with SSI-4produced a more durable anti-tumor response; complete tumor regressionand durable survival achieved in 28% of combination treated animals,where all animals in other treatment groups succumbed to tumor burden.SSI-4 monotherapy treated animals demonstrated improved median survivalof approximately 22% and 20% as compared to sham and PD-1 monotherapytreated groups, respectively.

FIGS. 23E-23F show that 4T1 TNBC tumors do not respond tomonotherapeutic PD1 blockade; combination of PD-1 blockade with SSI-4produced a statistically significant anti-tumor response; While notreatment groups achieved complete tumor regression, combination treatedanimals demonstrated improved median survival of approximately 75%,104%, and 40.8% as compared to sham, PD-1, and SSI-4 monotherapy treatedgroups, respectively.

FIG. 23G shows that SSI-4 monotherapy and combination therapy increasedthe number of tumor infiltrating macrophages in E0771-E2 HER2-positivetumors.

FIGS. 23H-23I shows that SSI-4, PD-1 monotherapy, and combinationtherapy had a deleterious effect on the intratumor population ofCD4-positive T regulatory cells; and SSI-4 monotherapy and combinationtherapy increased the number of cytotoxic CD8+ T cells in EO771-E2tumors.

FIGS. 24A-24B shows that depletion of CD8 T lymphocytes rescued theanti-tumor activity of the combination treatment in both TUBO andE0771-E2 HER2-positive tumors.

FIG. 25 shows that significant changes in the protein expression of thecheckpoint PD-L1 but not PD-L2 were observed in tumor-infiltratingdendritic and macrophage cells in E0771-E2 in response to each S SI-4monotherapy and combination therapy.

FIG. 26 shows that each CD4+ and CD8+ T lymphocytes demonstrateupregulation of protein expression of various checkpoints in response totherapy, including CTLA-4 and TIM3.

DETAILED DESCRIPTION

Recent advances in cancer immunotherapy indicate that hostresensitization to tumor presence can impart long-term survival benefitseven in patients with end-stage disease (1,2). Positive results havebeen observed in patients treated with checkpoint inhibitors such asantibody mediated anti-PD-1 blockade. Efficacy of this class of drugs,however, is limited to tumors considered to be immunogenic, exhibitingevidence of spontaneous T cell priming and immune cell infiltration (3).For poorly immunogenic tumors, existing platforms such as adoptive celltransfer or tumor vaccination aimed to achieve host resensitizationsuffer from low potency and inability to generate long-term immunememory (4).

While de novo lipogenesis is a normal physiological process, most normaltissues rely on exogenous uptake of free fatty acids (FA) from thebloodstream (5) including those with high proliferative capacity such ashematopoietic cells and intestinal epithelia (6). Contrary to this, manyaggressive cancers demonstrate increased fatty acid metabolism,establishing this phenomenon as a hallmark of oncogenesis (7,8). Assuch, targeting constituents of lipid biosynthesis has become a focusfor developing new anti-cancer therapies. SCD1 is an enzyme thatcatalyzes the de novo lipogenesis of Δ-9 monounsaturated fatty acids(MUFA) oleic acid (OA) and palmitoleic acid (PA), influencing a numberof cellular processes (9,10). Targeting SCD1 enzymatic activity inducesapoptosis in a variety of aggressive tumor models including kidney,liver, breast, lung, thyroid, and colon cancers (11-16).

The present disclosure describes that inhibition of de novo lipogenesisusing, e.g., SCD1 inhibitors, primes the tumor immune landscape towardsa pro-inflammatory phenotype and enhances the therapeutic benefit ofcheckpoint inhibitors such as anti-PD-1 agents.

In one general aspect, the present application provides a method ofincreasing the immunogenic susceptibility of a cell. In someembodiments, a cell with increased immunogenic susceptibility is able toprovoke a pro-inflammatory or immune response in vitro or in vivo, suchthat the cell is detected and neutralized by the immune system of thecell's host. In some embodiments, the neutralization comprises lysis ofthe cell, for example, by a cytolytic protein such as perforin, which isexpressed by the immune cells of the immune system of the host. Theimmune system may be an innate or an adaptive immune system. Suitableexamples of immune cells include natural killer (NK) cells,myeloid-derived suppressor cells (MDSC), red blood cells (RBC),thymocytes, megakaryocytes, innate lymphoid cells (ILC), granulocytes, Blymphocytes (B cells) and T lymphocytes (T cells). In some embodiments,the T-cell may be a natural killer T (NKT) cell, gamma delta T cell,regulatory T Cell (Treg), or helper T (Th) cell. In some embodiments,the T cell is a CD4+ T lymphocyte of a CD8+ T lymphocyte. In someembodiments, the immune cell comprises a checkpoint protein receptor,which suppresses the immune cell's inflammatory activity and protectsthe cells native to the host from autoimmunity. For example, when thecheckpoint protein receptor binds to a checkpoint receptor ligand on thesurface of the host's native cell (or a cancer cell), it can act as an“off-switch” that keeps the immune cell (e.g., T cell) from attackingthe native cell (or a cancer cell). Some immune cells need a checkpointprotein to be activated to start an immune response, for example, whenthe checkpoint protein is dissociated from its ligand. In someembodiments, the checkpoint protein is a programmed cell death protein-1(PD-1), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), or aT-cell immunoglobulin and mucin-domain containing-3 (TIM-3). In someembodiments, the checkpoint protein ligand is a programmeddeath-ligand-1 (PD-L1) or a programmed death-ligand-2 (PD-L2). In someembodiments, the checkpoint protein is LAG-3 (CD223) which is a cellsurface molecule expressed on activated T cells (See, e.g., Huard et al.Immunogenetics 39:213-217, 1994; Goldberg et al. Curr Top MicrobiolImmunol. 2011, 344, 269-78).

In some embodiments, the method of increasing immunogenic susceptibilityof a cell comprises contacting the cell with an effective amount of aninhibitor of de novo lipogenesis, or a pharmaceutically acceptable saltthereof. The contacting may occur in vitro (e.g., in a culture medium),or in vivo (e.g., by administering the inhibitor of de novo lipogenesisto the host of the cell). In some embodiments, prior to contacting thecell with the inhibitor of the de novo lipogenesis, the method comprisesselecting a poorly immunogenic cell (e.g., identifying a cell in need ofincreasing its immunogenic susceptibility). For example, the cell doesnot provoke a pro-inflammatory or immune response and may not beneutralized by an immune cell. In one example, a poorly immunogenic cellcomprises checkpoint protein ligand (e.g., PD-L1) which binds with thecheckpoint protein receptor on the surface of the immune cell and “turnsoff” the immune response. In some embodiments, contacting the cell withthe inhibitor of the de novo lipogenesis induces endoplasmic reticulum(ER) stress, which may provoke an adaptive immune response through theemission of immunostimulatory signals, or damage-associated molecularpatterns (DAMPs) such as heat shock proteins, or translocation ofcalreticulin (CRT) to the plasma membrane of the cell. In someembodiments, the ER stress leads to an increase in the immunogenicity ofthe cell.

In some embodiments, the cell is a cancer cell. Exemplary embodiments ofcancer cells are described herein.

Inhibitors of De Novo Lipogenesis

In some embodiments, the inhibitor of de novo lipogenesis is aninhibitor of fatty-acid synthesis. In some embodiments, the inhibitionof fatty acid synthesis subsequently inhibits the intracellularsynthesis of triglycerides, sphingolipids, glycolipids, phospholipids,lipoproteins and/or other fatty-acid containing molecules that influencemembrane fluidity, membrane raft formation and receptor clustering,second messenger signaling, fatty acid oxidation, energy storage, celldivision, inflammation, and a number of other biological functions. Insome embodiments, the inhibitor of de novo lipogenesis inhibits thesynthesis of unsaturated fatty acids. In some aspects of theseembodiments, the inhibitor of de novo lipogenesis inhibits the synthesisof monounsaturated fatty acids, such as a Δ-9 monounsaturated fatty acid(MUFA). In some embodiments, the inhibitor of de novo lipogenesisinhibits the synthesis of oleic acid (OA) and/or palmitoleic acid (PA).In some embodiments, the inhibitor of de novo lipogenesis is aninhibitor of stearoyl-CoA desaturase 1 (SCD1).

In some embodiments, the inhibitor of stearoyl-CoA desaturase 1 (SCD1)is a compound according to Formula (I):

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein:    -   R¹ is an unsubstituted C₁₋₆alkyl or C₁₋₆haloalkyl;    -   X is

Y is selected from:

-   -   m is 0 or 1;    -   n is 0, 1, or 2;    -   V is NR⁴ or O;    -   R², R³, and R⁴ are each independently H or an unsubstituted        C₁₋₆alkyl; and    -   Z is an unsubstituted aryl.

In some embodiments, the compound according to Formula (I) has thestructure of Formula (Ia):

or a pharmaceutically acceptable salt thereof.

In some embodiments, V is NR⁴. In some embodiments, V is NH. In someembodiments, V is O.

In some embodiments, X is

In some embodiments, Y is

In some embodiments, Y is

In some embodiments, Y is

In some embodiments, Z is selected from the group consisting of: phenyland naphthyl. For example, Z can be phenyl.

In some embodiments, m is 0. In some embodiments, m is 1.

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, n is 2.

In some embodiments, R¹ is an unsubstituted C₁₋₆alkyl. In someembodiments, R¹ is an unsubstituted C₁₋₃alkyl. For example, R¹ can beCH₃. In some embodiments, R¹ is a C₁₋₆haloalkyl. In some embodiments, R¹is a C₁₋₃haloalkyl. For example, R¹ can be CF₃.

In some embodiments, R² is an unsubstituted C₁₋₆alkyl. For example R²can be CH₃. In some embodiments, R² is H.

In some embodiments, R³ is an unsubstituted C₁₋₆alkyl. For example R³can be CH₃. In some embodiments, R³ is H.

In some embodiments, R⁴ is an unsubstituted C₁₋₆alkyl. For example R⁴can be CH₃. In some embodiments, R⁴ is H.

In some embodiments, R² is H; and R³ is CH₃.

Non-limiting examples of a compound according to Formula (I) and/orFormula (Ia) include:

N-Methyl-2-(2-oxo-2-{4-[2-(trifluoromethyl)benzoyl]piperazin-1-yl}ethoxy)benzamide;

2-(benzyloxy)-5-{[hydroxy({4-[2-(trifluoromethyl)benzoyl]piperazin-1-yl})methyl]amino}-1,2-dihydropyridin-2-ylium-1-ide;and

2-(benzyloxy)-4-({[hydroxy({4-[2-(trifluoromethyl)benzoyl]piperazin-1-yl})methyl]azanidyl}methyl)-1,2-dihydropyridin-2-ylium-1-ide,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of stearoyl-CoA desaturase 1 (SCD1)is a compound according to Formula (II):

-   -   or pharmaceutically acceptable salt thereof,    -   wherein    -   R¹ is halo;    -   X is —(C═O)NR⁴—;    -   Y is

-   -   R², R³, and R⁴ are each independently H or an unsubstituted        C₁₋₆alkyl.

In some embodiments, a compound according to Formula (II) has thestructure of Formula (IIa):

-   -   or pharmaceutically acceptable salt thereof.

In some embodiments, X is —(C═O)NR⁴—.

In some embodiments, Y is

In some embodiments, R¹ is Cl. In some embodiments, R¹ is F.

In some embodiments, R² is an unsubstituted C₁₋₆alkyl. For example R²can be CH₃. In some embodiments, R² is H.

In some embodiments, R³ is an unsubstituted C₁₋₆alkyl. For example R³can be CH₃. In some embodiments, R³ is H.

In some embodiments, R⁴ is an unsubstituted C₁₋₆alkyl. For example R⁴can be CH₃. In some embodiments, R⁴ is H.

In some embodiments, R² is H; and R³ is CH₃.

Non-limiting examples of a compound according to Formula (II) and/orFormula (IIa) include:

2-{[4-(2-Chlorophenoxy)piperidine-1-carbonyl]amino}-N-methylpyridine-4-carboxamide,

-   -   or pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of stearoyl-CoA desaturase 1 (SCD1)is:

-   -   or pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of stearoyl-CoA desaturase 1 (SCD1)is any one of the compounds described for example, in PCT applicationpublication No. WO 2016/141299, US publication No. 2013/0096181, or USpatent number 9,233,102, all of which are incorporated herein byreference in their entirety.

In some embodiments, the inhibitor of de novo lipogenesis is a fattyacid synthase (FASN) inhibitor (e.g., TVB-2640, fasnall, C 75, G 28UCM,GSK 2194069, Orlistat). The FASN inhibitor may inhibit the thioesterasedomain of fatty acid synthase. In some embodiments, the inhibitor of denovo lipogenesis is carboxylester lipase inhibitor. In some embodiments,the inhibitor of de novo lipogenesis is inhibitor of pyruvatedehydrogenase (PDH). In some embodiments, the inhibitor of de novolipogenesis is inhibitor of acetyl-CoA carboxylase. In some embodiments,the inhibitor of de novo lipogenesis is inhibitor of ATP-citrate lyase.

In another general aspect, the present application provides a method ofincreasing the immunogenic susceptibility of a tumor in a subject. Insome embodiments, the method comprises administering to the subject atherapeutically effective amount of an inhibitor of de novo lipogenesis,or a pharmaceutically acceptable salt thereof (e.g., any one ofinhibitors of de novo lipogenesis described herein). In someembodiments, the method comprises selecting a subject having a poorlyimmunogenic tumor.

In some embodiments, administration of the inhibitor of de novolipogenesis to the subject results in increased recruitment of apro-inflammatory antigen presenting cell (APC) into the tumormicroenvironment. Suitable examples of APCs are macrophages (MP) and/ordendritic cells (DC). In some embodiments, administration of theinhibitor of de novo lipogenesis to the subject increases the number ofintra-tumor DCs. In some embodiments, administration of the inhibitor ofde novo lipogenesis to the subject results in an increase in the numberof tumor-associated leukocytes in the subject of at least about 5%,about 10%, about 15%, about 20%, or about 25%. In some embodiments,antigen presenting cell activation augments infiltration and activationof the immune cells, such as T lymphocytes. In some embodiments,administration of the inhibitor of de novo lipogenesis to the subjectresults in production of cytolytic proteins such as perforin. Forexample, cytolytic proteins are produced by the immune cells responsiblefor tumor cell lysis during immunogenic cell death. In some embodiments,the immune cell is any one of the immune cells described herein, such asT lymphocytes (e.g., CD4+ or CD8+ T cells). In some embodiments,administration of the inhibitor of de novo lipogenesis to the subjectsensitizes the tumor for an immunotherapy or a checkpoint inhibitortherapy.

In another general aspect, the present application provides a method oftreating cancer (e.g., any one of cancers described herein) in asubject, the method comprising administering to the subject atherapeutically effective amount of an inhibitor of de novo lipogenesis(e.g., any one of the inhibitors described herein), or apharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of a checkpoint inhibitor (e.g., any one of checkpointinhibitors described herein), or a pharmaceutically acceptable saltthereof. In some embodiments, the subject is in need of the treatment(e.g., the subject is diagnosed with, or identified as having, acancer). In some embodiments, the method comprises selecting the subjectfor the combination treatment, for example, by determining that thecancer is poorly immunogenic, using any of the methods and/or kits knownin the art for analysis of tumor immunogenic susceptibility.

In some embodiments, the cancer is characterized in that the cancercells express a cell-surface checkpoint protein ligand (e.g., PD-L1).The ligand may bind with the checkpoint protein receptor (e.g., PD-1) onthe surface of an immune cell. In some embodiments, this binding leadsto inactivation of the immune cell, and the cancer cell remains intact(e.g., the cancer cell grows and proliferates despite attack by theimmune cell). In some embodiments, administration of an inhibitor of denovo lipogenesis to the subject induces and/or upregulates expression ofthe checkpoint protein ligands in the cancer cells of the subject. Inother embodiments, administration of an inhibitor of de novo lipogenesisto the subject does not change the levels of expression of thecheckpoint protein ligands in the cancer cells of the subject.

In some embodiments, a checkpoint inhibitor blocks the binding betweenthe checkpoint protein receptor of an immune cell and the checkpointprotein ligand of the cancer cell, thus activating the immune cell(“turning on the switch”). In some embodiments, administering acheckpoint inhibitor results in immunogenic death of the cancer cells.

In some embodiments, the cancer is associated with overexpression of anSCD1 protein, a SCD1 enzyme (e.g., “a SCD1-associated cancer”) (see,e.g., von Roemeling, C. A. et al. J. Clin. Endocrinol. Metab. (May 2015)100(5): E697-E709). The term “SCD1-associated cancer” as used hereinrefers to cancers associated with or having a dysregulation of a SCD1protein (SCD1 enzyme), or expression or activity or level of the same.

In some embodiment, the method comprises administering one or more(e.g., one, two, three, four, or more) of the inhibitors of de novolipogenesis; and administering one or more (e.g., one, two, three, four,or more) of the checkpoint inhibitors. In some embodiments, theinhibitor of de novo lipogenesis is administered with at least twocheckpoint inhibitors. In some embodiments, the inhibitor of de novolipogenesis is administered with at least three checkpoint inhibitors.

In some embodiments, the inhibitor of de novo lipogenesis and thecheckpoint inhibitor are admixed prior to administration (e.g., theinhibitor of de novo lipogenesis and the checkpoint inhibitor areadministered in a pharmaceutical composition) as described herein. Insome embodiments, the inhibitor of de novo lipogenesis and thecheckpoint inhibitor are administered concurrently. For example, theinhibitor of de novo lipogenesis is administered orally (e.g., in atablet or capsule); and the checkpoint inhibitor is simultaneouslyadministered intravenously. In some embodiments, the inhibitor of denovo lipogenesis and the checkpoint inhibitor are administeredsequentially, e.g., one of the therapeutic agents is administered priorto administration of the other therapeutic agent. For example, theinhibitor of de novo lipogenesis may be administered orally (e.g., in atablet or capsule) for a period of time (e.g. 1-10 days) prior tointravenous administration of the checkpoint inhibitor (e.g., one everythree months). In some embodiments, a method of treating cancer in asubject comprises administering to the subject SSI-4, or apharmaceutically acceptable salt thereof, and an anti-PD-1 antibody, ora pharmaceutically acceptable salt thereof

Checkpoint Inhibitors

In some embodiments, the checkpoint inhibitor is a small-molecule drug.Small molecule drugs are low molecular weight organic compounds(typically about 2000 daltons or less). In some embodiments, themolecular weight of the drug molecule is in the range from about 200 toabout 2000, from about 200 to about 1800, from about 200 to about 1600,from about 200 to about 1400, from about 200 to about 1200, from about200 to about 1000, from about 200 to about 800, from about 200 to about600 daltons, from about 300 to about 2000, from about 300 to about 1800,from about 300 to about 1600, from about 300 to about 1400, from about300 to about 1200, from about 300 to about 1000, from about 300 to about800, and/or from about 300 to about 600 daltons.

In some embodiments, the checkpoint inhibitor is a therapeutic proteinor a peptide, such as an antibody, a hormone, a transmembrane protein, agrowth factor, an enzyme, or a structural protein. In some embodiments,the checkpoint inhibitor is a biomolecule having a molecular weight of200 daltons or more produced by living organisms or cells, includinglarge polymeric molecules such as polypeptides, proteins, glycoproteins,polysaccharides, polynucleotides and nucleic acids, or analogs orderivatives of such molecules.

In some embodiments, the checkpoint inhibitor is an antibody, such as amonoclonal or a polyclonal antibody.

In some embodiments, the checkpoint inhibitor is selected form the groupconsisting of: a programmed cell death protein-1 (PD-1) inhibitor, aninhibitor of programmed death-ligand-1 (PD-L1) or programmeddeath-ligand-2 (PD-L2), an inhibitor of cytotoxicT-lymphocyte-associated protein-4 (CTLA-4), an inhibitor ofLymphocyte-activation gene 3 (LAG-3), an inhibitor of luster ofDifferentiation 47 (CD47), an inhibitor of Signal regulatory protein α(SIRP α) (e.g., TTI-621, OSE-172), and an inhibitor of T-cellimmunoglobulin and mucin-domain containing-3 (TIM-3). In someembodiments, the checkpoint inhibitor is a dual inhibitor of LAG-3 andPD-1 (e.g., MGD013). In some embodiments, the checkpoint inhibitor isselected form the group consisting of: an anti-PD-1 antibody (e.g.,pembrolizumab, nivolumab), an anti-PD-L1 antibody (e.g., atezolizumab),an anti-PD-L2 antibody, an anti-CTLA-4 antibody (e.g., ipilimumab), andan anti-TIM-3 antibody (e.g., TSR-022). In some embodiments, thecheckpoint inhibitor is selected form the group consisting of: PD-1inhibitor, PD-L1 inhibitor, PD-L2 inhibitor, TIM-1 inhibitor, TIM-3inhibitor, LAG-3 inhibitor, CTLA-4 inhibitor, CD-47 inhibitor, SIRPαinhibitor, and VISTA inhibitor. In some embodiments, the checkpointinhibitor is selected form the group consisting of: pembrolizumab,nivolumab, atezolizumab, ipilimumab, and TSR-022. In some embodiments,the checkpoint inhibitor is selected form the group consisting of:pembrolizumab, nivolumab, atezolizumab, ipilimumab, TSR-022, MGD013,TTI-621, OSE-172 and CA-170. In some embodiments, the method comprisesadministering to the subject at least two of: pembrolizumab, nivolumab,atezolizumab, ipilimumab, and TSR-022. In some embodiments, the methodcomprises administering to the subject at least two of: pembrolizumab,nivolumab, atezolizumab, ipilimumab, TSR-022, MGD013, TTI-621, OSE-172and CA-170. In some embodiments, the method comprises administering tothe subject at least three of: pembrolizumab, nivolumab, atezolizumab,ipilimumab, and TSR-022. In some embodiments, the method comprisesadministering to the subject at least three of: pembrolizumab,nivolumab, atezolizumab, ipilimumab, TSR-022, MGD013, TTI-621, OSE-172and CA-170. In some embodiments, the method comprises administering tothe subject pembrolizumab and nivolumab. In some embodiments, the methodcomprises administering to the subject pembrolizumab, nivolumab,atezolizumab, and ipilimumab. In some embodiments, the method comprisesadministering to the subject pembrolizumab, nivolumab, atezolizumab,ipilimumab, and TSR-022. In some embodiments, the method comprisesadministering to the subject pembrolizumab, nivolumab, atezolizumab,ipilimumab, TSR-022, MGD-013, TTI-621, OSE-172 and CA-170. In someembodiments, the checkpoint inhibitor is an antibody and is administeredto the subject intravenously. In some embodiments, the checkpointinhibitor is an antibody and is administered to the subject orally. Insome embodiments, the checkpoint inhibitor is a small-molecule drug thatis administered to the subject orally (e.g., CA-170). In someembodiments, when at least two checkpoint inhibitors are administered tothe subject, at least one checkpoint inhibitor is administered orally,and at least one checkpoint inhibitor is administered intravenously. Insome embodiments, all checkpoint inhibitors are administeredintravenously.

In some embodiments, the checkpoint inhibitor is any one of checkpointinhibitors described in Petrova et al., TTI-621 (SIRPαFc): ACD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Anti-TumorActivity and Minimal Erythrocyte Binding, Clinical Cancer Research, 2016(DOI: 10.1158/1078-0432.CCR-16-1700). In some embodiments, thecheckpoint inhibitor is inhibitor of CD47 (receptor) or SIRPalpha(ligand). In some embodiments, the checkpoint inhibitor is TTI-621, thatbinds to and neutralizes CD47, produced by Trillium Therapeutics Inc; orOSE-172, antagonist of SIRPα, produced by OSE Immunotherapeutics. Insome embodiments, the checkpoint inhibitor is an inhibitor of LAG-3(CD223) (e.g., MGD013, a dual inhibitor of LAG-3 and PD-1, manufacturedby MacroGenics). In some embodiments, the checkpoint inhibitor isV-domain Immunoglobulin Suppressor of T-cell Activation (VISTA)antagonist (e.g., CA-170 manufactured by Curtis Inc). In someembodiments, the checkpoint inhibitor selectively targets and inhibitboth PD-L1 and VISTA (e.g., CA-170). In some embodiments, the checkpointinhibitor is PD-L1/VISTA antagonist.

In some embodiments, an inhibitor of de novo lipogenesis (e.g., SCD1inhibitor such as SSI-4) modulates tumor immunity and is administeredwith an anti-PD-1 antibody and an anti-LAG3 antibody. In someembodiments, an inhibitor of de novo lipogenesis (e.g., SSI-4) modulatestumor immunity and synergizes with a checkpoint inhibitor (e.g.,anti-PD-1 antibody).

Cancer Cells

In some embodiments, the cancer is selected from the group consistingof: a kidney cancer, a liver cancer, a breast cancer, a lung cancer, apancreatic cancer, a bladder cancer, a colon cancer, a melanoma, athyroid cancer, an ovarian cancer, and a prostate cancer. The cancer maybe, for example, any one of the following cancers:

-   -   breast cancers, including, for example ER+ breast cancer, ER−        breast cancer, her2− breast cancer, her2+ breast cancer, stromal        tumors such as fibroadenomas, phyllodes tumors, and sarcomas,        and epithelial tumors such as large duct papillomas; carcinomas        of the breast including in situ (noninvasive) carcinoma that        includes ductal carcinoma in situ (including Paget's disease)        and lobular carcinoma in situ, and invasive (infiltrating)        carcinoma including, but not limited to, invasive ductal        carcinoma, invasive lobular carcinoma, medullary carcinoma,        colloid (mucinous) carcinoma, tubular carcinoma, and invasive        papillary carcinoma; and miscellaneous malignant neoplasms.        Further examples of breast cancers can include luminal A,        luminal B, basal A, basal B, and triple negative breast cancer,        which is estrogen receptor negative (ER−), progesterone receptor        negative, and her2 negative (her2−). In some embodiments, the        breast cancer may have a high risk Oncotype score;    -   hematopoietic cancers, including, for example, leukemia (acute        lymphocytic leukemia (ALL), acute lyelogenous leukemia (AML),        chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia        (CML), hairy cell leukemia), mature B cell neoplasms (small        lymphocytic lymphoma, B cell prolymphocytic leukemia,        lymphoplasmacytic lymphoma (such as Waldenström's        macroglobulinemia), splenic marginal zone lymphoma, plasma cell        myeloma, plasmacytoma, monoclonal immunoglobulin deposition        diseases, heavy chain diseases, extranodal marginal zone B cell        lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma        (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse B        cell lymphoma, diffuse large B cell lymphoma (DLBCL),        mediastinal (thymic) large B cell lymphoma, intravascular large        B cell lymphoma, primary effusion lymphoma and Burkitt        lymphoma/leukemia), mature T cell and natural killer (NK) cell        neoplasms (T cell prolymphocytic leukemia, T cell large granular        lymphocytic leukemia, aggressive NK cell leukemia, adult T cell        leukemia/lymphoma, extranodal NK/T cell lymphoma,        enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma,        blastic NK cell lymphoma, mycosis fungoides (Sézary syndrome),        primary cutaneous anaplastic large cell lymphoma, lymphomatoid        papulosis, angioimmunoblastic T cell lymphoma, unspecified        peripheral T cell lymphoma and anaplastic large cell lymphoma),        Hodgkin lymphoma (nodular sclerosis, mixed celluarity,        lymphocyte-rich, lymphocyte depleted or not depleted, nodular        lymphocyte-predominant), myeloma (multiple myeloma, indolent my        eloma, smoldering myeloma), chronic myeloproliferative disease,        myelodysplastic/myeloproliferative disease, myelodysplastic        syndromes, immunodeficiency-associated lymphoproliferative        disorders, histiocytic and dendritic cell neoplasms,        mastocytosis, chondrosarcoma, Ewing sarcoma, fibrosarcoma,        malignant giant cell tumor, and myeloma bone disease;    -   lung cancers, including, for example, bronchogenic carcinoma,        e.g., squamous cell, undifferentiated small cell, non-small cell        lung cancer (NSCLC), undifferentiated large cell, and        adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial        adenoma; sarcoma; lymphoma; chondromatous hamartoma; and        mesothelioma;    -   genitourinary tract cancers, including, for example, cancers of        the kidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma),        lymphoma, and leukemia; cancers of the bladder and urethra,        e.g., squamous cell carcinoma, transitional cell carcinoma, and        adenocarcinoma; cancers of the prostate, e.g., adenocarcinoma,        and sarcoma; cancer of the testis, e.g., seminoma, teratoma,        embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,        interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid        tumors, and lipoma;    -   liver cancers, including, for example, hepatoma, e.g.,        hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma;        angiosarcoma; hepatocellular adenoma; hepatobiliary carcinoma        (HCC), and hemangioma;    -   kidney (renal) cancers, including, for example, clear cell renal        cell carcinoma (ccRCC), papillary renal cell carcinoma,        chromophobe renal cell carcinoma, collecting duct renal cell        carcinoma, unclassified renal cell carcinoma, transitional cell        carcinoma, and renal sarcoma;    -   bladder cancers, including, for example, transitional cell        carcinoma, urothelial carcinoma, papillary carcinoma, flat        carcinoma, squamous cell carcinoma, adenocarcinoma, small-cell        carcinoma, and sarcoma;    -   gynecological cancers, including, for example, cancers of the        uterus, e.g., endometrial carcinoma; cancers of the cervix,        e.g., cervical carcinoma, and pre tumor cervical dysplasia;        cancers of the ovaries, e.g., ovarian carcinoma, including        serous cystadenocarcinoma, epithelial cancer, mucinous        cystadenocarcinoma, unclassified carcinoma, granulosa thecal        cell tumors, Sertoli Leydig cell tumors, dysgerminoma, and        malignant teratoma; cancers of the vulva, e.g., squamous cell        carcinoma, intraepithelial carcinoma, adenocarcinoma,        fibrosarcoma, and melanoma; cancers of the vagina, e.g., clear        cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and        embryonal rhabdomyosarcoma; and cancers of the fallopian tubes,        e.g., carcinoma;    -   skin cancers, including, for example, malignant melanoma, basal        cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles        dysplastic nevi, lipoma, angioma, dermatofibroma, keloids,        psoriasis;    -   thyroid cancers, including, for example, papillary thyroid        cancer, follicular thyroid cancer, anaplastic thyroid carcinoma,        and medullary thyroid cancer; and    -   adrenal gland cancers, including, for example, neuroblastoma.

In some embodiments, the cancer is a solid tumor.

Additional Anticancer Agents

In some embodiments, the method of treating cancer in a subject furthercomprises administering to the subject at least one additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis a pain relief agent (e.g., a nonsteroidal anti-inflammatory drug suchas celecoxib or rofecoxib), an antinausea agent, or an additionalanticancer agent (e.g., paclitaxel, docetaxel, doxorubicin,daunorubicin, epirubicin, fluorouracil, melphalan, cis-platin,carboplatin, cyclophosphamide, mitomycin, methotrexate, mitoxantrone,vinblastine, vincristine, ifosfamide, teniposide, etoposide, bleomycin,leucovorin, taxol, herceptin, avastin, cytarabine, dactinomycin,interferon alpha, streptozocin, prednisolone, irinotecan, sulindac,5-fluorouracil, capecitabine, oxaliplatin/5 FU, abiraterone, letrozole,5-aza/romidepsin, or procarbazine). In certain embodiments, theanticancer agent is paclitaxel or docetaxel. In other embodiments, theanticancer agent is cisplatin or irinotecan. In some embodiments, themethod of treating cancer in a subject further comprises administeringto the subject a cell carcinoma treatment. Examples of additionaloptional renal cell carcinoma treatments include, without limitation,treatment with Nexavar®, Sutent®, Torisel®, Afinitor® (everolimus),axitinib, pazopanib, levatinib, interleukin-2, and combinations thereof.In some embodiments, the method of treating cancer in a subject furthercomprises administering to the subject a proteasome inhibitor. Exemplaryproteasome inhibitors include lactacystin, bortezomib, dislfiram,salinosporamide A, carfilzomib, ONX0912, CEP-18770, MLN9708, epoxomicin,and MG132). Non-limiting examples of proteasome inhibitors includemarizomib (NPI-0052), bortezomib (Velcade®), and carfilzomib(Kyprolis®). Other suitable proteasome inhibitors can be found in U.S.Pat. Nos. 8,431,571; 8,357,683; 8,088,741; 8,080,576; 8,080,545;7,691,852; 7,687,456; 7,531,526; 7,109,323; 6,699,835; 6,548,668;6,297,217; 6,066,730, and published PCT applications WO 2011/123502; WO2010/036357; WO 2009/154737; WO 2009/051581; WO 2009/020448, each ofwhich is incorporated by reference in its entirety. In some embodiments,the combination of a compound provided herein (e.g., SSI-4) and aproteasome inhibitor have a synergistic effect on the treatment of thecancer. In some embodiments, the method of treating cancer in a subjectfurther comprises administering to the subject one or more multikinaseinhibitors (e.g., tyrosine kinase inhibitors, RAF kinase inhibitors,serine/threonine kinase inhibitors). In some embodiments, themultikinase inhibitor is sorafenib. In some embodiments, the combinationof a compound provided herein (e.g., SSI-4) and sorafenib have asynergistic effect on the treatment of the cancer. In some embodiments,the method of treating cancer in a subject further comprisesadministering to the subject an inhibitor of mammalian target ofrapamycin (mTor). Non-limiting examples of mTor inhibitors include:sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus (RAD001),ridaforolimus (AP-23573). In some embodiments, the method of treatingcancer in a subject further comprises administering to the subjectpacliltaxel and/or platin (cisplatin, carboplatin, or oxaliplatin) forthe treatment of ovarian cancer.

In some embodiments, the following standard of care drugs can becombined with an inhibitor of de novo lipogenesis and a checkpointinhibitor for the following cancers: Lung—paclitaxel, nivolumab,ceritinib, afatinib; Colon—capecitabine; Breast; Metastaticbreast—capecitabine, paclitaxel, and/or gemcitabine; Hormonallyresponsive breast—aromatase inhibitors such as letrazole and/orantiestrogens such as tamoxifen; HER2 positive—Her2 inhibitors such astrastuzumab; palbociclib, ado-trastuzumab emtansine;Melanoma—temozolomide, and/or BRAF inhibitors, pembrolizumab, nivolumab,pomalidomide, dabrafenib; Prostate—androgen receptor inhibitors such asabiraterone; Bladder—gemcitabine and/or paclitaxel; Thyroid—paclitaxel,cisplatin, a proteasome inhibitor, sorafenib, lenvatinib;Pancreatic—gemcitabine; Liver—sorafenib; Mantle celllymphoma—bortezomib; Multiple myeloma—panobinostat; Relapsed and/orrefractory—carfilzomib, bortezomib and/or an immunomodulatory agent suchas dexamethasone.

Pharmaceutically Acceptable Salts

In some embodiments, the present application provides a pharmaceuticallyacceptable salt of any one of the compounds disclosed herein (e.g., aninhibitor of de novo lipogenesis compound, a checkpoint inhibitorcompound, or an additional therapeutic agent). In some embodiments, asalt of any one of the compounds disclosed herein is formed between anacid and a basic group of the compound, such as an amino functionalgroup, or a base and an acidic group of the compound, such as a carboxylfunctional group. According to another embodiment, the compound is apharmaceutically acceptable acid addition salt.

In some embodiments, acids commonly employed to form pharmaceuticallyacceptable salts of the compounds disclosed herein include inorganicacids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid and phosphoric acid, as well as organicacids such as para-toluenesulfonic acid, salicylic acid, tartaric acid,bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid,gluconic acid, glucuronic acid, formic acid, glutamic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lacticacid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid,succinic acid, citric acid, benzoic acid and acetic acid, as well asrelated inorganic and organic acids. Such pharmaceutically acceptablesalts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caprate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate,xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate and other salts. In one embodiment,pharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, andespecially those formed with organic acids such as maleic acid.

In some embodiments, bases commonly employed to form pharmaceuticallyacceptable salts of the compounds disclosed herein include hydroxides ofalkali metals, including sodium, potassium, and lithium; hydroxides ofalkaline earth metals such as calcium and magnesium; hydroxides of othermetals, such as aluminum and zinc; ammonia, organic amines such asunsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines,dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine;diethylamine; triethylamine; mono-, bis-, ortris-(2-OH—(C1-C6)-alkylamine), such asN,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine;pyrrolidine; and amino acids such as arginine, lysine, and the like.

Compositions, Formulations, Dosages, Routes of Administration

In some embodiments, the present application provides pharmaceuticalcompositions comprising an inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.

In some embodiments, the present application provides pharmaceuticalcompositions comprising a checkpoint inhibitor, or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier.

In some embodiments, the present application provides pharmaceuticalcompositions comprising an inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof; a checkpoint inhibitor, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

The carrier(s) are “acceptable” in the sense of being compatible withthe other ingredients of the formulation and, in the case of apharmaceutically acceptable carrier, not deleterious to the recipientthereof in an amount used in the medicament.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of the present applicationinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

If required, the solubility and bioavailability of the compounds of thepresent application in pharmaceutical compositions may be enhanced bymethods well-known in the art. One method includes the use of lipidexcipients in the formulation. See “Oral Lipid-Based Formulations:Enhancing the Bioavailability of Poorly Water-Soluble Drugs (Drugs andthe Pharmaceutical Sciences),” David J. Hauss, ed. Informa Healthcare,2007; and “Role of Lipid Excipients in Modifying Oral and ParenteralDrug Delivery: Basic Principles and Biological Examples,” Kishor M.Wasan, ed. Wiley-Interscience, 2006.

Another known method of enhancing bioavailability is the use of anamorphous form of a compound of the present application optionallyformulated with a poloxamer, such as LUTROL™ and PLURONIC™ (BASFCorporation), or block copolymers of ethylene oxide and propylene oxide.See U.S. Pat. No. 7,014,866; and United States patent publications20060094744 and 20060079502, all of which are hereby incorporated byreference in their entireties.

The pharmaceutical compositions of the present application include thosesuitable for oral, rectal, nasal, topical (including buccal andsublingual), vaginal, parenteral, or intraperitoneal (includingsubcutaneous, intramuscular, intravenous and intradermal)administration. In certain embodiments, the compound of the formulaeherein is administered transdermally (e.g., using a transdermal patch oriontophoretic techniques). Other formulations may conveniently bepresented in unit dosage form, e.g., tablets, sustained releasecapsules, and in liposomes, and may be prepared by any methods wellknown in the art of pharmacy. See, for example, Remington: The Scienceand Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore, MD(20th ed. 2000).

Solutions or suspensions used for parenteral, intravenous, intradermal,intraocular or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution (e.g., 0.9% saline solution), dextrose solution (e.g., 5%)dextrose solution), fixed oils, polyethylene glycols (e.g., PEG400),glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. The parenteral preparation can beenclosed in ampoules, disposable syringes or multiple dose vials made ofglass or plastic.

Such preparative methods include the step of bringing into associationwith the molecule to be administered ingredients such as the carrierthat constitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredients with liquid carriers, liposomes orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

In some embodiments, an inhibitor of de novo lipogenesis is administeredorally. Compositions of the present application suitable for oraladministration may be presented as discrete units such as capsules,sachets, or tablets each containing a predetermined amount of the activeingredient; a powder or granules; a solution or a suspension in anaqueous liquid or a non-aqueous liquid; an oil-in-water liquid emulsion;a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc.Soft gelatin capsules can be useful for containing such suspensions,which may beneficially increase the rate of compound absorption.

In the case of tablets for oral use, carriers that are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried cornstarch. When aqueoussuspensions are administered orally, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring and/or coloring agents may be added.

Compositions suitable for oral administration include lozengescomprising the ingredients in a flavored basis, usually sucrose andacacia or tragacanth; and pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain antioxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.In some embodiments, a checkpoint inhibitor is administeredintravenously (e.g., by injection or infusion).

Such injection solutions may be in the form, for example, of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant.

The pharmaceutical compositions of the present application may beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound with a suitablenon-irritating excipient which is solid at room temperature but liquidat the rectal temperature and therefore will melt in the rectum torelease the active components. Such materials include, but are notlimited to, cocoa butter, beeswax, and polyethylene glycols.

The pharmaceutical compositions of the present application may beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art. See, for example, U.S. Pat. No. 6,803,031.

Topical administration of the pharmaceutical compositions of the presentapplication is especially useful when the desired treatment involvesareas or organs readily accessible by topical application.

The topical compositions of the present disclosure can be prepared andused in the form of an aerosol spray, cream, emulsion, solid, liquid,dispersion, foam, oil, gel, hydrogel, lotion, mousse, ointment, powder,patch, pomade, solution, pump spray, stick, towelette, soap, or otherforms commonly employed in the art of topical administration and/orcosmetic and skin care formulation. The topical compositions can be inan emulsion form.

Application of the subject therapeutics may be local, so as to beadministered at the site of interest. Various techniques can be used forproviding the subject compositions at the site of interest, such asinjection, use of catheters, trocars, projectiles, pluronic gel, stents,sustained drug release polymers or other device which provides forinternal access.

In the pharmaceutical compositions of the present application, aninhibitor of de novo lipogenesis or a checkpoint inhibitor is present inan effective amount (e.g., a therapeutically effective amount).

Effective doses will also vary, as recognized by those skilled in theart, depending on the diseases treated, the severity of the disease, theroute of administration, the sex, age and general health condition ofthe subject, excipient usage, the possibility of co-usage with othertherapeutic treatments such as use of other agents and the judgment ofthe treating physician.

In some embodiments, a therapeutically effective amount of an inhibitorof de novo lipogenesis is from about 10 to about 1000 mg/m², from about20 to about 900 mg/m², from about 30 to about 800 mg/m², from about 40to about 700 mg/m², from about 50 to about 800 mg/m², from about 50 toabout 150 mg/m², from about 60 to about 600 mg/m², from about 70 toabout 500 mg/m², or from about 100 to about 500 mg/m². 50-150 mg/m²

In some embodiments, a therapeutically effective amount of an inhibitorof de novo lipogenesis is from about 5 to about 300 mg/kg, from about 10mg/kg to about 250 mg/kg, from about 10 mg/kg to about 200 mg/kg, orfrom about 20 mg/kg to about 150 mg/kg. In some embodiments, atherapeutically effective amount of an inhibitor of de novo lipogenesisis from about 50 mg/kg to about 500 mg/kg, from about 50 mg/kg to about400 mg/kg, from about 50 mg/kg to about 300 mg/kg, from about 90 mg/kgto about 280 mg/kg, from about 100 mg/kg to about 250 mg/kg, from about130 mg/kg to about 230 mg/kg, from about 150 mg/kg to about 200 mg/kg,or from about 200 mg/kg to about 250 mg/kg. Exemplary doses includeabout 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, about 50mg/kg, about 75 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150mg/kg, about 180 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250mg/kg, about 275 mg/kg, about 300 mg/kg, or about 350 mg/kg. Any ofthese doses may be administered once daily, twice daily or three timesdaily, or once a week, once a month or once every three months. In someembodiments, an inhibitor of de novo lipogenesis is administered orallyonce daily by a tablet or capsule.

In some embodiments, a therapeutically effective amount of a checkpointinhibitor is from about 1 to about 15 mg/m², or from about 1 to about 15mg/m². In some embodiments, a therapeutically effective amount of acheckpoint inhibitor is from about 10 μg/dose to about 1000 μg/dose,from about 20 μg/dose to about 800 μg/dose, from about 30 μg/dose toabout 600 μg/dose, from about 40 μg/dose to about 500 μg/dose, fromabout 50 μg/dose to about 400 μg/dose, from about 60 μg/dose to about300 μg/dose, from about 70 μg/dose to about 200 μg/dose, or from about80 μg/dose to about 120 μg/dose.

In some embodiments, a therapeutically effective amount of a checkpointinhibitor is from about 0.05 mg/kg to about 100 mg/kg, from about 0.1mg/kg to about 75 mg/kg, from about 0.2 mg/kg to about 50 mg/kg, fromabout 0.5 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30mg/kg, from about 0.5 mg/kg to about 20 mg/kg, from about 0.5 mg/kg toabout 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1 mg/kgto about 15 mg/kg, from about 0.5 mg/kg to about 5 mg/kg, or from about1 mg/kg to about 5 mg/kg. Exemplary doses include about 0.5 mg/kg, about1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg,about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10mg/kg. Any of these doses may be administered once daily, twice daily orthree times daily, or once a week, once a month, or once every threemonths.

In some embodiments, a method of treating cancer in a subject comprisesadministering to the subject from about 100 mg/kg to about 250 mg/kg ofan inhibitor of de novo lipogenesis, or a pharmaceutically acceptablesalt thereof, and from about 1 mg/kg to about 10 mg/kg a checkpointinhibitor, or a pharmaceutically acceptable salt thereof. In someembodiments, a method of treating cancer in a subject comprisesadministering to the subject from about 130 mg/kg to about 230 mg/kg ofan inhibitor of de novo lipogenesis, or a pharmaceutically acceptablesalt thereof, and from about 0.5 mg/kg to about 5 mg/kg a checkpointinhibitor, or a pharmaceutically acceptable salt thereof. In someembodiments, a method of treating cancer in a subject comprisesadministering to the subject from about 200 mg/kg to about 250 mg/kg ofan inhibitor of de novo lipogenesis, or a pharmaceutically acceptablesalt thereof, and from about 1 mg/kg to about 15 mg/kg a checkpointinhibitor, or a pharmaceutically acceptable salt thereof. In someaspects of the aforementioned embodiments, the inhibitor of de novolipogenesis is administered orally (e.g., as a tablet or capsule), andthe checkpoint inhibitor is administered intravenously (e.g., byinfusion).

In some embodiments, an inhibitor of de novo lipogenesis and acheckpoint inhibitor are administered such that the molar ratio of theinhibitor of de novo lipogenesis, or a pharmaceutically acceptable saltthereof, to the checkpoint inhibitor, or a pharmaceutically acceptablesalt thereof, is from about 1000:1 to about 1:50, from about 900:1 toabout 1:40, from about 800:1 to about 1:30, from about 700:1 to about1:20, from about 500:1 to about 1:10, from about 200:1 to about 1:5, orfrom about 150:1 to about 1:3. In some embodiments, an inhibitor of denovo lipogenesis and a checkpoint inhibitor are administered such thatthe molar ratio of the inhibitor of de novo lipogenesis, or apharmaceutically acceptable salt thereof, to the checkpoint inhibitor,or a pharmaceutically acceptable salt thereof, is from about 3:1 toabout 1:3, or from about 2:1 to about 1:2. In some aspects of theseembodiments, the molar ratio is about 10:1, about 9:1, about 8:1, about7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1,about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about1:8, about 1:9, or about 1:10.

In some embodiments, a method of treating cancer in a subject comprisesadministering to the subject SSI-4, or a pharmaceutically acceptablesalt thereof, and an anti-PD-1 antibody, or a pharmaceuticallyacceptable salt thereof. In some aspects of these embodiments, SSI-4, ora pharmaceutically acceptable salt thereof, is administered in an amountfrom about 130 mg/kg to about 230 mg/kg; and an anti-PD-1 antibody, or apharmaceutically acceptable salt thereof, is administered in an amountfrom about 0.5 mg/kg to about 5 mg/kg. In other aspects of theseembodiments, SSI-4, or a pharmaceutically acceptable salt thereof, isadministered in an amount from about 200 mg/kg to about 250 mg/kg; andan anti-PD-1 antibody, or a pharmaceutically acceptable salt thereof, isadministered in an amount from about 1 mg/kg to about 15 mg/kg. Infurther aspects of these embodiments, the anti-PD-1 antibody ispembrolizumab or nivolumab. In yet further aspects of these embodiments,SSI-4, or a pharmaceutically acceptable salt thereof, is administeredorally (e.g., as a tablet or capsule), and the anti-PD-1 antibody isadministered intravenously (e.g., by infusion).

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment of cancer, which include one or morecontainers containing an inhibitor of de novo lipogenesis, a checkpointinhibitor, or a pharmaceutical composition comprising same. Such kitscan further include, if desired, one or more of various conventionalpharmaceutical kit components, such as, for example, containers with oneor more pharmaceutically acceptable carriers, additional containers,etc., as will be readily apparent to those skilled in the art.Instructions, either as inserts or as labels, indicating quantities ofthe components to be administered, guidelines for administration, and/orguidelines for mixing the components, can also be included in the kit.

Definitions

As used herein, the term “cell” is meant to refer to a cell that is invitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can bepart of a tissue sample excised from an organism such as a mammal. Insome embodiments, an in vitro cell can be a cell in a cell culture. Insome embodiments, an in vivo cell is a cell living in an organism suchas a mammal.

As used herein, the term “individual”, “patient”, or “subject” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” refers to the amount of active compound orpharmaceutical agent that elicits the biological or medicinal responsein a tissue, system, animal, individual or human that is being sought bya researcher, veterinarian, medical doctor or other clinician.

As used herein the term “treating” or “treatment” refers to 1)inhibiting the disease; for example, inhibiting a disease, condition ordisorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,arresting further development of the pathology and/or symptomatology),or 2) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., reversing the pathology and/or symptomatology).

The term, “compound”, as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted. Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.,hydrates and solvates).

The term “halo” refers to fluoro, chloro, bromo or iodo.

The term “alkyl” refers to a straight or branched chain alkyl group,having from 1-20 carbon atoms. The alkyl is unsubstituted unlessotherwise indicated. Illustrative of the alkyl group include the methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl,3-methylbutyl, 2,2-dimethylpropyl, 1,1-dimethylpropyl, hexyl,1-methylpentyl, 4-methylpentyl, heptyl, 1-methylhexyl, 2-methylhexyl,5-methylhexyl, 3-ethylpentyl, octyl, 2-methylheptyl, 6-methylheptyl,2-ethylhexyl, 2-ethyl-3-methylpentyl, 3-ethyl-2-methylpentyl, nonyl,2-methyloctyl, 7-methyloctyl, 4-ethylheptyl, 3-ethyl-2-methylhexyl,2-ethyl-1-methylhexyl, decyl, 2-methylnonyl, 8-methylnonyl,5-ethyloctyl, 3-ethyl-2-methylheptyl, 3,3-diethylhexyl, undecyl,2-methyldecyl, 9-methyldecyl, 4-ethylnonyl, 3,5-dimethylnonyl,3-propyloctyl, 5-ethyl-4-methyloctyl, 1-pentylhexyl, dodecyl,1-methylundecyl, 10-methylundecyl, 3-ethyldecyl, 5-propylnonyl,3,5-diethyloctyl, tridecyl, 11-methyldodecyl, 7-ethylundecyl,4-propyldecyl, 5-ethyl-3-methyldecyl, 3-pentyloctyl, tetradecyl,12-methyltridecyl, 8-ethyldodecyl, 6-propylundecyl, 4-butyldecyl,2-pentylnonyl, pentadecyl, 13-methyltetradecyl, 10-ethyltridecyl,7-propyldodecyl, 5-ethyl-3-methyldodecyl, 4-pentyldecyl, 1-hexylnonyl,hexadecyl, 14-methylpentadecyl, 6-ethyltetradecyl, 4-propyltridecyl,2-butyldodecyl, heptadecyl, 15-methylhexadecyl, 7-ethylpentadecyl,3-propyltetradecyl, 5-pentyldodecyl, octadecyl, 16-methylheptadecyl,5-propylpentadecyl, nonadecyl, 17-methyloctadecyl, 4-ethylheptadecyl,icosyl, 18-methylnonadecyl, 3-ethyloctadecyl, henicosyl, docosinyl,tricosinyl, tetracosinyl and pentacosinyl groups.

The term “C_(x-y) alkyl” refers to an alkyl group between x and y carbonatoms in size. For example, C1-8 alkyl refers to an alkyl of 1 to 8carbon atoms.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedunsubstituted single-ring aromatic groups in which each atom of the ringis carbon. The term “aryl” also includes polycyclic ring systems havingtwo cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is aromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. The aryl group may beoptionally substituted where indicated. Aryl groups include benzene,naphthalene, tetralin, and the like.

The term “haloalkyl” refers to an alkyl group that is substituted withone or more (e.g., 1, 2, 3, 4, or 5) halo substituents. The group isotherwise unsubstituted unless as indicated. Examples includechloroethyl, chloromethyl, difluoromethyl, trifluoromethyl, and thelike.

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EXAMPLES Example 1-SCD1 is Correlated with Poor Patient Outcomes inHER2-Positive Breast Cancer

The effects of SCD1 inhibition in clear cell renal cell carcinoma(ccRCC) were investigated. Gene array and ancillary pathway signatureanalysis of SCD1 inhibitor treated ccRCC cells revealed profoundalterations in acute phase inflammatory signaling (FIG. 1 a ),suggestive that this compound may influence tumor cell inflammatoryreprogramming. SCD1 inhibitors could behave as immunosensitizing agentsin cancer. To determine the effects of SSI-4 in tumor immunity, anappropriate immune-competent model of carcinogenesis was identified.SCD1 mRNA expression is increased in breast cancer. The online platformGene expression-based Outcome for Breast cancer Online (GOBO) for SCD1expression among different subtypes of breast cancer was searched andmaximal SCD1 over-expression in HER2 enriched breast cancer wasdetermined (FIG. 1 b ). Using this platform patient samples werestratified by subtype and the relationship between patient overallsurvival and SCD1 expression was examined. While high SCD1 expression iscorrelated with a mild decrease in patient overall survival (OS) in allbreast cancer (FIG. 2 c ), HER2-enriched patients with elevated SCD1expression had a marked decrease in OS as compared to SCD1-low patients(FIG. 2 d ). In the following examples, the role of SSI-4 mediated tumorimmunogenicity in HER2-enriched breast cancer was shown using 4 murinetumor models: TUBO, E0771-E2, N202, and MMTV-neu.

Example 2-SSI-4 Induces Translocation of Calreticulin to the PlasmaMembrane

SSI-4 induces ER stress in tumor cells. Treatment of TUBO, E0771-E2, andN202 tumor cells with SSI-4 (10-1000 nM) resulted in activation of ERstress as shown by increased levels of phosphorylated eukaryotictranslation initiation factor 2 alpha (eIF2a) at serine51 and DNA damageinducible transcript 3 (CHOP, DDIT3) (FIG. 1 e ). ER stress can provokea therapeutically relevant adaptive immune response against malignantcells through the emission of immunostimulatory signals, ordamage-associated molecular patterns (DAMPs) such as heat shock proteinsand translocation of calreticulin (CRT) to the plasma membrane (17). Inparticular, phosphorylation of eIF2a has been reported to mediate CRTtranslocation (20) which is correlated with the induction of immunogeniccell death (ICD) and favorable disease outcome in a variety ofmalignancies (17). SSI-4 potently upregulates plasma membrane expressionof CRT at doses as low as 10 nM, as measured by flow cytometry on livetumor cells following a 48 hour treatment (FIG. 1 f ).

Example 3—SSI-4 Treatment Instigates Adaptive Immunity In Vitro

CRT behaves as a pro-phagocytic signal. The effect of SSI-4 treatment ontumor cell phagocytosis by bone marrow-derived macrophages (BMDM) invitro was determined. SSI-4 (1000 nM) induced the highest level ofphagocytosis in both TUBO and E0771-E2 cells (5 and 13%, respectively)(FIG. 2 a ). Neutralization of CRT using a blocking antibody was able tosignificantly abrogate this effect in both cell models, suggesting thatSSI-4 mediated phagocytosis is due in part to CRT translocation (FIG. 2a ). Downstream activation of T lymphocytes is dependent on thesuccessful maturation and presentation of antigen by antigen-presentingcells such as macrophages (Woo 2015). SSI-4 driven activation of BMDMsin vitro was evaluated by measuring antigen presentation of chickenovalbumin (cOva) by MHC class I receptors on BMDM after co-culturingthem with cOva-expressing tumor cells. SSI-4 treatment (100 nM)significantly enhanced antigen presentation in both TUBO (8.5%) andE0771-E2 (5%) cells, and this effect was inhibited with adjuvant CRTneutralization (FIG. 2 b ). CD8+ T lymphocyte activation was evaluatedin vitro using splenic T cells derived from OT-I transgenic mice whichrecognize ovalbumin residues 257-264 in the context of H2Kb.Proliferation and interferon gamma (IFNγ) production was measured in Tlymphocytes co-cultured with both cOva-expressing tumor cells and BMDMtreated with SSI-4. In addition, in order to evaluate whether theeffects of SSI-4 on T lymphocyte activation were direct or mediatedthrough a tumor-specific mechanism, concurrent analysis in SSI-4 treatedT lymphocytes alone and T lymphocytes co-cultured with BMDM only wasperformed. SSI-4 (1000 nM) was able to potently induce CD8+ T lymphocyteproliferation as well as IFNγ production in both E0771-E2 and MMTV-neutriple cultures (FIG. 2 c-d ). CRT neutralization was able to abrogatethese effects in MMTV-neu cells, while a mild (n.s.) decrease in IFNγproduction was observed in E0771-E2 cells (FIG. 2 c-d ). SSI-4 drivenCRT translocation is a contributor to these results. No significantchanges in T lymphocyte proliferation or IFNγ production were observedin alone or T lymphocyte-BMDM co-cultures, e.g., activation occurs in atumor-specific antigen-mediated manner (FIG. 2 c-d ).

Example 4—SSI-4 Demonstrates Anti-Tumor Activity in Immune-CompetentMice

SSI-4 mediated immunomodulation in vivo was evaluated. TUBO cells wereinjected orthotopically into the mammary fat pad of BALB/c mice. Animalsreceived either sham or SSI-4 (180 mg/kg) orally (continuous) when tumorburden reached 50-100 mm³. SSI-4 treated animals demonstrated slowertumor progression, with markedly smaller tumor sizes recorded at 30 daysafter onset of therapy when control animals reached endpoint (FIG. 3 a). An appreciable increase in overall survival was seen in SSI-4 treatedmice (FIG. 3 b ). Tumor tissue was harvested 14 days after treatmentonset for analysis. H&E staining of tumor sections did not revealconspicuous changes in overall tissue morphology between sham and SSI-4treatment groups (FIG. 3 c ), however a significant reduction in theproliferative capacity of SSI-4 treated tumors was recorded viadecreased nuclear Ki67 staining (FIG. 3 c ). In addition, a significantincrease in cleaved caspase 3 (CC3) staining, indicative of enhancedtumor apoptosis was noted in the SSI-4 group (FIG. 3 d ).

Example 5—SSI-4 Treatment Enhances Immunogenicity of Poorly-ImmunogenicBreast Cancer

As SSI-4 treatment of tumor cells enhances the antigen presentingcapabilities of APCs in vitro, tumor-associated recruitment ofprofessional phagocytes in vivo including macrophages (MP) and dendriticcells (DC) was evaluated. The ratio of tumor leukocyte recruitment bycomparing the ratio of cluster of differentiation 45 antigen (CD45)positive to negative cells sorted from dissociated tumor tissue withincontrol and SSI-4 treated mice was determined. Results indicated a 10%increase in the number of tumor-associated leukocytes within SSI-4treated animals (FIG. 3 f ). Next, tumor sections were stained forF4/80, a macrophage (MP) marker. SSI-4 treated tumors demonstratedexpanded expression of MPs, as well as increased intra-tumor penetrationof these cells toward the tumor core (FIG. 3 g ). To determineintra-tumor dendritic cell infiltration, tumors were dissociated andanalyzed via flow cytometry for DCs based on CD45⁺MHCII⁺CD11c^(hi)expression. A significant increase in the number of intra-tumor DCswithin SSI-4 treated animals as compared to both control treated tumorsand normal, non-tumor bearing mice (FIG. 3 h ) was observed. Astumor-associated macrophage (TAM) cells can be correlated with either atumor-suppressive or pro-inflammatory phenotype, we sorted thispopulation identified by MHCII⁺CD11c^(lo)F4/80⁺ based on thepolarization markers interleukin-1β (IL-1β) and IL-10, comparing theexpression profiles from both treatment groups as well as mammary tissueextracted from non-tumor bearing mice. While no changes were observedbetween sham and SSI-4 treated TAM for IL-10, an immunosuppressivecytokine, a significant increase in IL-1β, a cytokine produced by maturemacrophages and indicative of pro-inflammatory activation, was observedin response to SSI-4 treatment (FIG. 3 i ). These data support thatSSI-4 promotes the recruitment of pro-inflammatory antigen presentingcells (APCs) into the tumor microenvironment in vivo.

SSI-4 mediated APC activation could augment T lymphocyte infiltrationand activation. IHC analysis of tumor sections for T lymphocytedistribution revealed a significant increase in the number of both CD4+and CD8+ tumor-infiltrating populations (FIG. 4 a-b ). A significantincrease in perforin, a cytolytic protein produced by activatedcytotoxic T lymphocytes responsible for tumor cell lysis during ICD, wasobserved in SSI-4 treated tumors (FIG. 4 c ). The maturation status of Tlymphocytes from CD3+ cells isolated from the spleen of treated animalsby comparing the ratio of CD44 to CD62L expression in either CD4 or CD8T-cells was examined. SSI-4 treatment produced a robust induction ofmemory and effector T-cells among both CD4 and CD8 positive populations;along with a concomitant decrease in naïve T-cell numbers (FIG. 4 d-e ).The activation status of T lymphocytes isolated from digested tumors wasalso assessed. The number of effector CD4 and CD8 T lymphocytesidentified by IFNγ, a cytokine predominantly produced by activatedcytotoxic lymphocytes, was markedly increased in both CD4 and CD8+populations with SSI-4 (FIG. 4 f ). Granzyme B-positive CD8 T-cells werealso enriched in SSI-4 treated tumors (FIG. 4 f ), another marker foractivated CD8+ T lymphocytes. In parallel, SSI-4 treatment correspondedwith a profound decrease in the number of CD4 and CD8 positiveintra-tumor regulatory T lymphocytes (Treg), characterized by dual CD25and FoxP3 expression (FIG. 4 f ). Collectively, these data indicate thatSSI-4 is able to bolster intratumor TIL recruitment and maturation, andpromoting ICD in HER2 positive breast cancer cells.

Example 6—SSI-4 Augments PD-1 Blockade Mediated Anti-Tumor T CellImmunity

Materials and methods: The checkpoint inhibitor used was a mouseanti-PD-1 that was purchased from BioXCell (catalog #BE0146). Thecheckpoint inhibitor was administered at 100 μg/dose (5 mg/kg) byintraperitoneal (IP) injection.

As tumor progression invariably occurred in SSI-4 treated mice, possiblemechanisms of resistance were explored. Tumor-mediated upregulation ofimmunosuppressive checkpoints promotes T lymphocyte anergism, thusenhancing tumor resistance to immunotherapy (Woo 2015). IHC analysis oftumor sections revealed that SSI-4 treatment induces programmed deathligand-1 (PD-L1) expression in TUBO tumor bearing mice (FIG. 4 g ). Tonegate the effects of PD-L1 upregulation, the combination of PD-1antibody-mediated blockade, the receptor for PD-L1, and SSI-4, wastested. In E0771-E2 tumor bearing mice, anti-PD-1 therapy produced nosurvival benefit, and tumor burden was comparable to placebo,demonstrating that these tumors do not respond to monotherapeutic PD-1blockade (FIG. 5 b-c ). The combination of PD-1 blockade with SSI-4produced a more durable anti-tumor response as compared to bothmonotherapies and control treated animals, as evidenced by decreasedtumor burden in this group once the placebo group reached endpointparameters (FIG. 5 b ). Median survival in the combination groupincreased by approximately 45% compared to both placebo and PD-1 alone,and 20% as compared to SSI-4 monotherapy (FIG. 5 c ). Tumor dissociationand isolation and characterization of T-lymphocytes revealed asignificant increase in effector CD8+ cytotoxic T lymphocytes inresponse to SSI-4 and combination therapy (FIG. 5 d-e ), suggesting thatthese cells may play more of a prominent role in mediating anti-tumorresponsiveness of the treatment. PD-1 monotherapy and combinationtherapy also appeared to have a deleterious effect on the intratumorpopulation of T regulatory cells, which are known to contribute to tumorresistance to immunotherapy (FIG. 5 f ). To determine whether theanti-tumor effect observed was dependent on the activity of cytotoxicCD8-positive T lymphocytes, combination treatment was repeated in Balb/cmice bearing TUBO tumors in the presence of CD8-blocking antibody.Depletion of CD8 T lymphocytes rescued the anti-tumor activity of thecombination treatment (FIG. 5 g ). Successful splenic depletion of CD8T-cells in animals receiving CD-8 blockade (FIG. 5 h ) was confirmed.

To better understand mechanisms of drug resistance to combinationtherapy, E0771-E2 tumor tissue was harvested from treated mice, and theexpression level of known checkpoint proteins was evaluated onintratumor dendritic cells, macrophages, and T lymphocytes. Results showthat macrophages are the predominant resident leukocyte in these tumors,where dendritic cells represent less than 1% of all CD45% cells (FIG. 6a ). A significant influx of macrophages into the tumor was observed inresponse to either SSI-4 or combination therapy (FIG. 6 a ). Nosignificant changes in the protein expression of the checkpoints PD-L1or PD-L2 were observed in dendritic cells (FIG. 6 b ). Macrophagesshowed increased PD-L1 in response to both SSI-4 and combination therapy(FIG. 6 c ). Both CD4 and CD8 positive T lymphocytes demonstrateupregulation of protein expression of various checkpoints in response totherapy, including CTLA-4 and TIM3 (FIG. 6 d-e ). These findings suggestthat tumors respond to SSI-4 and combination therapy by upregulatingother known checkpoints in an effort to mount tumor-resistance.Combination therapy with SSI-4 that includes a cocktail of checkpointinhibitors such as anti-PD-L1, anti-CTLA4, and/or anti-TIM3 may providea more durable anti-tumor response.

FIGS. 5G-5H, 6A-6E: Tumor-infiltrating (TI) leukocytes and lymphocyteswere assessed by multicolor flow cytometry on dissociated treated tumortissue extracted from E0771-E2 bearing mice on day 27 of the study. 6A:The % of dendritic cells (DC) vs. macrophages (mac) that make upTI-Leukocytes was determined, and demonstrate that mac are thepredominant TI-leuk in these tumors. 6B: The expression of thecheckpoints PD-L1 and PD-L2 on either DC or Mac in response to therapydemonstrate that SSI-4 (and combinatorial therapy) strongly induce PD-L1expression on macrophages. The expression of the checkpoints PD-1,CTLA-4, and TIM-3 was assessed in either 6D: CD4-expressing or 6E:CD8-expressing T lymphocytes, and show upregulation of both CTLA-4 andTIM-3 in response to SSI-4 and/or combination therapy. 5G: To determinewhether anti-tumor activity of SSI-4/PD-1 combination therapy isdependent on cytotoxic CD8 T lymphocyte activity, CD8 depletion studieswere performed in mice receiving either placebo or combination therapy.Combination treated mice bearing E0771-E2 tumors demonstratedsignificant reduction in tumor burden as compared to placebo mice, andthis was reversed in the presence of CD8 depletion. 5H: Flow cytometricanalysis was performed on splenic T cells isolated from animals in (5G),and confirm successful depletion of CD8 T cells within animals receivingCD8 blockade.

Without being bound by a particular theory, it is believed that usingseveral poorly immunogenic models of orthotopic HER2 breast cancer, theexamples describes herein show that SCD1 inhibitors such as SSI-4enhance tumor antigen presenting cell (APC) recruitment and maturation,as well as T cell priming both in vitro and in vivo. In monotherapytreated immunocompetent murine models, SCD1 inhibitors such as SSI-4 ledto a significant reduction in tumor burden and increase in survival ascompared to controls. Tissue analysis revealed tumor infiltration ofeffector T lymphocytes, and reduction of anti-inflammatory T regulatory(Treg) cells, redolent of a T-cell inflamed phenotype. The resultsfurther show that SCD1 inhibitors such as SSI-4 are able to sensitizeresistant tumors to programed death-1 (PD-1) inhibition, resulting inreduced tumor burden and significantly prolonged survival. Thesefindings demonstrate that SCD1 inhibitors such as SSI-4 modulate tumorimmunity and synergize with the checkpoint inhibitors such as PD-1blockers.

OTHER EMBODIMENTS

It is to be understood that while the present application has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the present application, which is defined by the scope of theappended claims. Other aspects, advantages, and modifications are withinthe scope of the following claims.

1-111. (canceled)
 112. A method of treating cancer in a subject, themethod comprising administering to the subject in need thereof: (i) atherapeutically effective amount of a compound of formula:

or a pharmaceutically acceptable salt thereof, and (ii) atherapeutically effective amount of at least two checkpoint inhibitorsindependently selected from: an antibody of programmed cell deathprotein-1 (PD-1); an antibody of programmed death-ligand-1 (PD-L1); anantibody of programmed death-ligand-2 (PD-L2); an antibody of cytotoxicT-lymphocyte-associated protein-4 (CTLA-4); an antibody ofLymphocyte-activation gene 3 (LAG-3); an antibody of Cluster ofDifferentiation 47 (CD47); an antibody of Signal regulatory protein α(SIRP α); an antibody of T-cell immunoglobulin and mucin-domaincontaining molecule (TIM-3) or (TIM-1); an antibody of V-domainimmunoglobulin (Ig) suppressor of T cell activation (VISTA); and a dualantibody of LAG-3 and PD-1.
 113. The method of claim 112, wherein the atleast two checkpoint inhibitors are independently selected from: ananti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, ananti-CTLA-4 antibody, and an anti-TIM-3 antibody.
 114. The method ofclaim 112, wherein the compound, or a pharmaceutically acceptable saltthereof, is administered to the subject with two checkpoint inhibitors.115. The method of claim 114, wherein the compound, or apharmaceutically acceptable salt thereof, is administered to the subjectwith an anti-CTLA-4 antibody and an anti-TIM-3 antibody.
 116. The methodof claim 114, wherein the compound, or a pharmaceutically acceptablesalt thereof, is administered to the subject with an anti-CTLA-4antibody and an anti-PD-1 antibody.
 117. The method of claim 114,wherein the compound, or a pharmaceutically acceptable salt thereof, isadministered to the subject with an anti-TIM-3 antibody and an anti-PD-1antibody.
 118. The method of claim 114, wherein the two checkpointinhibitors are independently selected from: pembrolizumab, nivolumab,atezolizumab, ipilimumab, and TSR-022.
 119. The method of claim 112,wherein the compound, or a pharmaceutically acceptable salt thereof, isadministered with at least three checkpoint inhibitors.
 120. The methodof claim 119, wherein the compound, or a pharmaceutically acceptablesalt thereof, is administered with three checkpoint inhibitors.
 121. Themethod of claim 120 wherein the compound, or a pharmaceuticallyacceptable salt thereof, is administered to the subject with ananti-CTLA-4 antibody, an anti-TIM-3 antibody, and an anti-PD-1 antibody.122. The method of claim 120, wherein the three checkpoint inhibitorsare selected from: pembrolizumab, nivolumab, atezolizumab, ipilimumab,and TSR-022.
 123. The method of claim 112, wherein the compound, or apharmaceutically acceptable salt thereof, and the at least twocheckpoint inhibitors, are administered concurrently.
 124. The method ofclaim 112, wherein the compound, or a pharmaceutically acceptable saltthereof, and the at least two checkpoint inhibitors, are administeredsequentially.
 125. The method of claim 112, wherein the compound, or apharmaceutically acceptable salt thereof, is administered orally. 126.The method of claim 112, wherein each checkpoint inhibitor isadministered intravenously.
 127. The method of claim 112, wherein thecompound, or a pharmaceutically acceptable salt thereof, is administeredin an amount from about 200 mg/kg to about 250 mg/kg.
 128. The method ofclaim 112, wherein the cancer is selected from the group consisting of:a kidney cancer, a liver cancer, a breast cancer, a lung cancer, apancreatic cancer, a bladder cancer, a colon cancer, a melanoma, athyroid cancer, an ovarian cancer, and a prostate cancer.